CN113005116B - Microbial preparation and application thereof in aspect of water purification - Google Patents
Microbial preparation and application thereof in aspect of water purification Download PDFInfo
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- CN113005116B CN113005116B CN202110124473.2A CN202110124473A CN113005116B CN 113005116 B CN113005116 B CN 113005116B CN 202110124473 A CN202110124473 A CN 202110124473A CN 113005116 B CN113005116 B CN 113005116B
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 94
- 230000000813 microbial effect Effects 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 238000000746 purification Methods 0.000 title claims abstract description 23
- 240000006055 Dacrydium cupressinum Species 0.000 claims abstract description 68
- 235000018782 Dacrydium cupressinum Nutrition 0.000 claims abstract description 68
- 235000013697 Pinus resinosa Nutrition 0.000 claims abstract description 68
- 241000894006 Bacteria Species 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 39
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000001179 sorption measurement Methods 0.000 claims abstract description 18
- 239000010802 sludge Substances 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 15
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 11
- 238000004132 cross linking Methods 0.000 claims abstract description 11
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 46
- 229910052698 phosphorus Inorganic materials 0.000 claims description 46
- 239000011574 phosphorus Substances 0.000 claims description 46
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 238000011049 filling Methods 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- 238000000967 suction filtration Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 238000012546 transfer Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007952 growth promoter Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 239000003086 colorant Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- RLJMLMKIBZAXJO-UHFFFAOYSA-N lead nitrate Chemical compound [O-][N+](=O)O[Pb]O[N+]([O-])=O RLJMLMKIBZAXJO-UHFFFAOYSA-N 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229910001220 stainless steel Inorganic materials 0.000 claims description 4
- 239000010935 stainless steel Substances 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 1
- 239000001110 calcium chloride Substances 0.000 claims 1
- 229910001628 calcium chloride Inorganic materials 0.000 claims 1
- 235000011148 calcium chloride Nutrition 0.000 claims 1
- 238000009360 aquaculture Methods 0.000 abstract description 9
- 244000144974 aquaculture Species 0.000 abstract description 9
- 240000007263 Pinus koraiensis Species 0.000 abstract description 7
- 235000011615 Pinus koraiensis Nutrition 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 7
- 230000006872 improvement Effects 0.000 abstract description 5
- 230000004048 modification Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 5
- 230000007797 corrosion Effects 0.000 abstract description 3
- 238000005260 corrosion Methods 0.000 abstract description 3
- 231100000956 nontoxicity Toxicity 0.000 abstract 1
- 241000881711 Acipenser sturio Species 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 229910001385 heavy metal Inorganic materials 0.000 description 9
- 241000195493 Cryptophyta Species 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 238000011065 in-situ storage Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 231100000719 pollutant Toxicity 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000012851 eutrophication Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237536 Mytilus edulis Species 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000020638 mussel Nutrition 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 230000001546 nitrifying effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/04—Arrangements for treating water specially adapted to receptacles for live fish
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/28—Treatment of water, waste water, or sewage by sorption
- C02F1/286—Treatment of water, waste water, or sewage by sorption using natural organic sorbents or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/30—Aerobic and anaerobic processes
- C02F3/308—Biological phosphorus removal
-
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/20—Bacteria; Culture media therefor
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- C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
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- C02F2101/105—Phosphorus compounds
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- C02F2305/06—Nutrients for stimulating the growth of microorganisms
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Abstract
The invention discloses a microbial preparation and application thereof in water purification, which relates to the field of aquaculture water quality improvement, wherein target strains are selected from eutrophic water and activated sludge to obtain aerobic phosphorus-accumulating bacteria, then the aerobic phosphorus-accumulating bacteria are subjected to seed expansion culture and prepared into constant volume liquid, the constant volume liquid is modified by citric acid and red pine nut shells, and then the constant volume liquid is placed into the constant volume liquid for adsorption, mixed with polyethylene glycol and stirred, and then dripped into a water tank containing CaCl 2 Solidifying and crosslinking, washing, freeze-drying and removing water to obtain the microbial preparation. The invention has the beneficial effects that: the polyethylene glycol is added to the red pine nut shells through modification, so that the strength of the modified red pine nut shells is effectively improved, and the corrosion to microorganisms is improvedCorrosion resistance, no toxicity to organisms, and good biocompatibility; and the high-valued application of the Korean pine nut shells is realized.
Description
Technical Field
The invention relates to the field of aquaculture water quality improvement, in particular to a microbial preparation and application thereof in water purification.
Background
China is a major country for producing the red pine nuts, the export of the red pine nuts is the first place in the world, the export of the red pine nuts is about 40% of the total world transaction amount, the waste in the processing production process of the red pine nut shells as the red pine nuts accounts for about 70% of the whole cones, and the red pine nuts cannot be fully utilized, so that the resource waste is caused, and the environment is damaged.
In the last twenty years, the intensive aquaculture industry rapidly develops at home and abroad, brings great economic benefits, however, many aquaculture households lack sufficient knowledge of the ecological problem severity of the aquaculture industry, and lack theoretical guidance of a method for solving the problem, in order to unilaterally increase the aquaculture yield of unit water, the stocking density is often excessively increased, excessive feeding and fertilization are carried out, on one hand, organic matters, nitrogen and phosphorus in the water are greatly increased, the eutrophication of the water is caused, the oxygen content of the water environment is rapidly reduced, algae are rapidly propagated, the algae are malodorous and toxic, the concentration of dissolved oxygen in the water is reduced, the transparency of the water is reduced, and the water quality is deteriorated; on the other hand, excessive organic matters in the water body and excrement of the cultured organisms are decomposed to further consume dissolved oxygen in the water body, so that the water body environment is in an anoxic state, algae propagation is further accelerated, sunlight is shielded by the algae, aquatic plants die due to the obstruction of photosynthesis, pathogenic bacteria and spoilage bacteria in the water body environment are bred and nutrient substances of plants such as nitrogen and phosphorus are released, and are used by the algae, so that vicious circle is caused and cultured organisms in the water body are endangered, the biodiversity in the cultured water body is reduced, and the culture of high-quality aquatic products is limited by water quality and bait organisms.
According to the relation of the algae on the nitrogen and phosphorus requirements, the phosphorus requirements are more important, and the growth and propagation of the algae are particularly limited by the phosphorus, so that the reduction of the phosphorus content in the water body is one of the important means for purifying the eutrophication of the culture water body.
The future aquaculture industry development is limited by water resources, land resources, environmental protection, energy utilization, aquatic product quality and other important factors, so that the aquaculture water quality and the natural water body are subjected to in-situ improvement and removal of harmful substances, the aquaculture water resources are recycled on the basis of ensuring the safety of the aquatic products, the natural and artificial dual recovery of the ecological environment is realized, the environment is effectively protected, the circular economy is developed, and the sustainable development is realized. Therefore, in-situ improvement of water quality and removal of harmful substances are widely studied in recent years, chinese patent CN110078224A adopts a micro-energy consumption microorganism purification system to purify water body, chinese patent CN208151154U adopts a sewage circulation treatment system to purify water body, but large occupied space is needed, equipment investment cost is high and in-situ removal cannot be carried out; chinese patent CN109052834A is prepared by uniformly mixing bacillus subtilis, nitrifying bacteria and denitrifying bacteria according to a certain proportion to purify water, but heavy metals cannot be treated; chinese patent CN107583618A adopts sodium alginate/mussel shell powder microspheres to purify water, but has the defect of being incapable of removing phosphorus pollution.
Disclosure of Invention
Aiming at the problem that the prior art cannot fully utilize the Korean pine nuts; the large occupied space is needed, the equipment investment cost is high, and the equipment cannot be cleaned in situ; the invention discloses a microbial preparation which can only realize the problems of phosphorus pollution removal or heavy metal physical adsorption, and is prepared by the following steps:
inoculating aerobic phosphorus accumulating bacteria into a synthetic culture medium, and culturing for a period of time to obtain an aerobic phosphorus accumulating bacteria liquid;
step two, removing bacterial liquid supernatant through centrifugation, and fixing the volume by using normal saline to obtain constant volume liquid;
and thirdly, weighing the modified red pine nut shells, and placing the modified red pine nut shells into constant volume liquid for adsorption to obtain mixed liquid.
Step four, dripping the mixed liquid into CaCl-containing liquid by an injector with the needle diameter of 6-8mm 2 Solidifying into balls, cross-linking for a period of time, washing with physiological saline, freeze-drying in a freeze dryer, and taking out freeze-dried particles to obtain the required microbial preparation. The microbial preparation is specifically an aerobic phosphorus accumulating bacterium immobilized particle.
Preferably, the components and the concentrations of the synthetic medium in the first step are respectively as follows: 20g/L of activated sludge, 15g/L, naCl g/L of trypsin and 5g/L of yeast extract, and the solvent is an eutrophic water body.
Preferably, in step one, at 25℃200 r.min -1 Anaerobic and aerobic continuous culture is carried out for 2-3 days under the condition of (1) to obtain aerobic phosphorus accumulating bacteria liquid.
Preferably, the centrifugal rotation speed in the second step is 5000 r.min -1 The time is 20min, and the volume is fixed to 300mL.
Preferably, in the third step, the modified red pine nut shells are weighed and put into constant volume liquid for adsorption, and then are mixed with polyethylene glycol, and the mixture is obtained after uniform stirring.
Preferably, the weight of the modified red pine nut shell is 150g, the adsorption time is 1h, the polyethylene glycol volume is 200mL, and the mass fraction is 10%.
Preferably, the modified red pine nut shell in the third step is modified by the following steps:
step 3.1, accurately weighing 150g of red pine nut shells, crushing the red pine nut shells to a size of 4-6mm, placing the red pine nut shells in a 3L beaker, and adding 1.8L of 0.5 mol.L -1 After mixing the citric acid solutions of (2) at room temperature at 600 r.min -1 Stirring at a speed of 30-40min, transferring into a stainless steel plate,drying in a blast drying oven at 50-55deg.C for 24-36h, and reacting at 120deg.C for 90min;
step 3.2, after cooling the modified red pine nut shells to room temperature, washing the modified red pine nut shells with deionized water until the washing liquid does not cause turbidity of a 0.1M lead nitrate solution any more, and carrying out suction filtration by using suction filtration equipment;
and 3.3, drying the modified red pine nut shells to constant weight at 60 ℃, and placing the dried modified red pine nut shells in a dryer for standby.
Preferably, the CaCl-containing material of step four 2 Is 300mL in volume of CaCl 2 The mass fraction of (2%), the crosslinking time was 24 hours, and the lyophilization temperature was 5-10 ℃.
Preferably, the aerobic phosphorus accumulating bacteria in the first step are screened by adopting the following steps:
step 1.1, taking 80mL of eutrophic water body and 20g of activated sludge, adding 20mL of growth promoter, mixing, and heating at 25 ℃ for 200 r.min -1 After 3 hours of culture under the condition of (2) and standing for 0.5 hour;
step 1.2, collecting 10mL of supernatant, inoculating onto bacterial culture medium, and culturing at 25deg.C and 200r.min -1 Is continued to be cultured for 5 days under the condition of (2);
step 1.3, transferring 5mL of culture solution, and keeping the culture medium and culture conditions unchanged;
step 1.4, after transfer culture for 5-6 times, continuously carrying out transfer culture for 3 times on single bacterial colonies with different colors and forms in a separation culture medium containing eutrophic water and activated sludge by adopting a streak inoculation method, and repeatedly separating and purifying to obtain aerobic phosphorus accumulating bacteria single bacterial colonies;
step 1.5, respectively inoculating the obtained aerobic phosphorus accumulating bacteria single colonies into 100mL of synthetic medium for anaerobic and aerobic continuous culture for 2-3 days, measuring the phosphorus removal efficiency of each strain, and selecting the strain with the best phosphorus removal effect as a target strain.
Preferably, in step 1.5, the components and the concentrations thereof in the synthetic medium are respectively: 20g/L of activated sludge, 15g/L, naCl g/L of trypsin and 5g/L of yeast extract, wherein the solvent is eutrophic water, the culture temperature is 25 ℃, and the stirring rate is 200 r.min -1 。
The invention also provides an application of the microbial preparation, and the microbial preparation is placed in a purifying unit which can be suspended in a water body.
Preferably, the purification unit comprises a floating bed for providing buoyancy, a containing device for filling the microbial preparation and a lifting rope for controlling the lowering depth of the containing device, one end of the lifting rope is telescopically bound on the floating bed, and the other end of the lifting rope is bound on the containing device.
Preferably, the accommodating device comprises an accommodating device cover with a through hole on the surface and an accommodating device main body with an inner wall and an outer wall, wherein the inner wall and the outer wall of the accommodating device main body are respectively provided with wall holes, and the inner wall and the outer wall divide the interior of the accommodating device main body into an inner cavity for filling the solid growth promoting agent and an outer cavity for filling the microbial agent.
The beneficial effects are that:
the technical scheme of the invention has the following beneficial effects:
(1) The polyethylene glycol is added to the red pine nut shells through modification, so that the strength of the modified red pine nut shells is effectively improved, the corrosion resistance to microorganisms is improved, and the modified red pine nut shells are nontoxic to organisms and have good biocompatibility;
(2) Through crosslinking, the aerobic phosphorus accumulating bacteria can be better fixed on the immobilized particles, so that the flowing water is prevented from scattering bacterial groups, the aerobic phosphorus accumulating bacteria are beneficial to exerting the bacterial group advantage, the water purifying efficiency is improved, and the physical adsorption capacity of the modified red pine nut shells is also improved;
(3) The microbial preparation, namely the aerobic phosphorus accumulating bacteria immobilized particles, can adsorb humic substances, nutritive salts and excretions of aquatic animals in water, improve the utilization efficiency of the aerobic phosphorus accumulating bacteria group lodged in the immobilized particles to water, excretions and organic substances in the humic substances, improve the phosphorus uptake and phosphorus removal rate of the aerobic phosphorus accumulating bacteria group, promote the growth and propagation of the aerobic phosphorus accumulating bacteria group, facilitate the full play of the advantages of the aerobic phosphorus accumulating bacteria group, reduce the phosphorus content in the water, reduce the contents of the excretions and the humic substances in the water, limit the growth and propagation of algae, spoilage bacteria and pathogenic bacteria in the water, adsorb heavy metals released in the nutritive salts and sludge, and avoid secondary pollution when purifying the water;
(4) The microbial preparation is placed in the outer cavity of the accommodating device in the purifying unit, the inner cavity of the accommodating device is filled with the solid growth promoting agent, so that aerobic phosphorus bacteria in the immobilized particles can grow and reproduce rapidly by utilizing the solid growth promoting agent in the inner cavity, the advantage of forming a flora by the aerobic phosphorus bacteria is facilitated, in addition, the growth promoting agent which is partially diffused outside the accommodating device can activate indigenous microorganisms with pollutant degradation capability in water environment, by utilizing a biological competition eliminating method, the indigenous microorganism place with pollutant degradation capability is improved, the indigenous microorganisms are rapidly reproduced, community structures are optimized, the oxygen content in water environment is improved, the self-cleaning capability of the environment is improved, the pollutants are degraded or converted into harmless substances in situ, the purifying unit can adapt to the purification of harsher and complex water bodies, and in addition, the purifying unit has small occupied space and low equipment investment cost and is convenient to use, and in-situ treatment can be realized.
(5) The Korean pine nut shells adopted in the invention can realize the high-valued application of the waste Korean pine nut shells to a certain extent, change waste into valuable, and avoid wasting resources and polluting the environment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a perspective view of a preferred purification unit of the present invention;
FIG. 2 is a bottom view of a preferred housing device cover of the present invention;
fig. 3 is a bottom view of the main body of the preferred accommodating device of the present invention.
In the figure:
1-a purification unit; 11-floating bed; 12-accommodating devices; 121-housing a device cover;
122-housing a device body; 13-a lifting rope; 2-wall holes; 3-through holes; 4-inner wall;
5-an outer wall; 6-inner cavity; 7-outer cavity.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the examples of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, provided in the examples, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
In the embodiment, target strains are selected from eutrophic water and activated sludge to obtain aerobic phosphorus-accumulating bacteria, the aerobic phosphorus-accumulating bacteria are subjected to seed expansion culture and prepared into constant volume liquid, citric acid and Korean pine nut shells are used for modification to obtain modified Korean pine nut shells, the modified Korean pine nut shells are placed into the constant volume liquid for adsorption, and then are mixed with polyethylene glycol, stirred uniformly and then dripped into a water tank containing CaCl 2 Solidifying and crosslinking, washing with physiological saline, freeze-drying to remove water to obtain the required microbial preparation. The preparation method comprises the following steps:
step one, taking 80mL of eutrophic water body and 20g of activated sludge, adding 20mL of growth promoter, mixing, and heating at 25 ℃ for 200 r.min -1 After 3 hours of culture under the condition of (2) and standing for 0.5 hour; collecting 10mL of supernatant, inoculating onto bacterial culture medium, and culturing at 25deg.C and 200r.min -1 Continuously culturing for 5 days under the condition of (1) taking 5mL of culture solution for transferring, keeping the culture medium and culture condition unchanged, and waiting for transferring cultureAfter 5-6 times of cultivation, single bacterial colonies with different colors and forms are continuously subjected to 3 times of transfer cultivation by adopting a streak inoculation method in a separation culture medium containing eutrophic water and activated sludge, and repeated separation and purification are carried out to obtain aerobic phosphorus accumulating bacteria single bacterial colonies; inoculating the obtained aerobic polyphosphoric bacteria single colony into each component and the concentration thereof are respectively as follows: 20g/L of activated sludge, 15g/L, naCl g/L of trypsin and 5g/L of yeast extract, wherein the solvent is eutrophic water, and the pH is in 100mL of synthetic culture medium with natural pH at 25 ℃ and 200 r.min -1 After anaerobic and aerobic continuous culture is carried out for 2-3 days under the condition of that, the dephosphorization efficiency of each strain is measured, the strain with the best dephosphorization effect is selected as the target strain, the target strain is inoculated into 500mL of the synthetic culture medium, and the temperature is 25 ℃ and 200 r.min -1 Anaerobic and aerobic continuous culture is carried out for 2-3 days under the condition of (2) to obtain aerobic phosphorus accumulating bacteria liquid;
step two, rotating at 5000 r.min -1 Centrifuging for 20min, pouring out supernatant, and fixing volume to 300mL by using physiological saline with mass fraction of 0.9% to obtain constant volume liquid;
step three, accurately weighing 150g of red pine nut shells, crushing the red pine nut shells to a size of 4-6mm, placing the red pine nut shells into a 3L beaker, and adding 1.8L of 0.5 mol.L -1 After mixing the citric acid solutions of (2) at room temperature at 600 r.min -1 Stirring at the speed of 30-40min, transferring into a stainless steel plate, drying at 50-55deg.C in a forced air drying oven for 24-36h, and heating to 120deg.C for reaction for 90min; cooling the modified red pine nut shells to room temperature, washing with deionized water until the washing liquid does not cause turbidity of the 0.1M lead nitrate solution, and carrying out suction filtration by using suction filtration equipment; drying the modified red pine nut shells to constant weight at 60 ℃, and placing the dried modified red pine nut shells in a dryer for standby; 150g of modified red pine nut shells in a dryer are taken out and put into the constant volume liquid, and are mixed with 200mL of polyethylene glycol with the mass fraction of 10% after being adsorbed for 1h, and are stirred uniformly to obtain a mixed liquid.
Step four, dripping the mixed liquid to CaCl with the volume of 300mL and the mass fraction of 2% by using an injector with the needle diameter of 6-8mm 2 Solidifying into balls, cross-linking for 24 hr, washing with 0.9 wt% physiological saline, freeze-drying at 5-10deg.C, and taking outLyophilizing the granule to obtain the desired microbial preparation. The microbial preparation is specifically an aerobic phosphorus accumulating bacterium immobilized particle.
As shown in fig. 1, the present embodiment also provides the use of a microbial preparation placed in a purification unit 1 that can be suspended in a body of water.
As a preferred embodiment, the purification unit 1 includes a floating bed 11 for providing buoyancy, a receiving device 12 for filling the microbial agent, and a hanging rope 13 for controlling a lowering depth of the receiving device 12, one end of the hanging rope 13 is telescopically bound to the floating bed 11, and the other end of the hanging rope 13 is bound to the receiving device 12.
As shown in fig. 1, 2 and 3, as a preferred embodiment, the container 12 includes a container cover 121 having a through hole 3 formed on a surface thereof and a container body 122 having an inner wall 4 and an outer wall 5, wherein the inner wall 4 and the outer wall 5 of the container body 122 are provided with wall holes 2, and the inner wall 4 and the outer wall 5 divide an interior of the container body 122 into an inner cavity 6 for filling with a solid growth promoter and an outer cavity 7 for filling with the microbial agent.
The beneficial effects of the microbial preparation obtained by the technical scheme of the present embodiment will be further described below by way of several sets of examples and comparative examples.
Embodiment one:
the screening of the target strain is carried out according to the embodiment, and the specific steps are as follows:
taking 80mL of eutrophic water and 20g of activated sludge, adding 20mL of growth promoter, mixing, and heating at 25deg.C for 200 r.min -1 After 3 hours of culture under the condition of (2) and standing for 0.5 hour; collecting 10mL of supernatant, inoculating onto bacterial culture medium, and culturing at 25deg.C and 200r.min -1 Continuously culturing for 5 days under the condition of (1) taking 5mL of culture solution for transferring, keeping the culture medium and the culture condition unchanged, continuously performing transfer culture for 3 times on single bacterial colonies with different colors and forms in a separation culture medium containing eutrophic water bodies and activated sludge by adopting a streak inoculation method after transfer culture for 6 times, repeatedly separating and purifying to obtain aerobic phosphorus accumulating bacteria single bacterial colonies, and performing the process of culturingThe selected aerobic phosphorus accumulating bacteria system is named as X-i (wherein i refers to any natural number).
Inoculating X-i into each component and the concentration thereof are respectively as follows: 20g/L of activated sludge, 15g/L, naCl g/L of trypsin and 5g/L of yeast extract, wherein the solvent is eutrophic water, and the pH is in 100mL of synthetic culture medium with natural pH at 25 ℃ and 200 r.min -1 The anaerobic and aerobic continuous culture was carried out under the conditions of 1d, 2d and 3d for 3 days, and the biomass and TP removal rate of each strain were measured, wherein the biomass was measured as OD600, and the specific data are shown in Table 1 and Table 2.
Embodiment two:
the embodiment is to prepare a microbial preparation, namely to prepare aerobic polyphosphoric bacteria immobilized particles, and the specific steps are as follows:
step one, culturing by using the X-9 obtained in the first embodiment in the embodiment to obtain an aerobic phosphorus accumulating bacterial liquid;
step two, rotating at 5000 r.min -1 Centrifuging for 20min, pouring out supernatant, and fixing volume to 300mL by using physiological saline with mass fraction of 0.9% to obtain constant volume liquid;
step three, accurately weighing 150g of red pine nut shells, crushing the red pine nut shells to a size of 4-6mm, placing the red pine nut shells into a 3L beaker, and adding 1.8L of 0.5 mol.L -1 After mixing the citric acid solutions of (2) at room temperature at 600 r.min -1 Stirring for 40min, transferring into a stainless steel plate, baking at 53 ℃ for 36h in a forced air drying oven, and raising the temperature to 120 ℃ for reaction for 90min; cooling the modified red pine nut shells to room temperature, washing with deionized water until the washing liquid does not cause turbidity of the 0.1M lead nitrate solution, and carrying out suction filtration by using suction filtration equipment; drying the modified red pine nut shells to constant weight at 60 ℃, and placing the dried modified red pine nut shells in a dryer for standby; 150g of modified red pine nut shells in a dryer are taken out and put into the constant volume liquid, and are mixed with 200mL of polyethylene glycol with the mass fraction of 10% after being adsorbed for 1h, and are stirred uniformly to obtain a mixed liquid.
Step four, dripping the mixed liquid to CaCl with the volume of 300mL and the mass fraction of 2% by using an injector with the needle diameter of 6-8mm 2 In the saturated boric acid solution, curing and balling, and after crosslinking for 24 hours, using the mass fraction of 0.9%Is put into a freeze dryer to be freeze-dried at 5 ℃ and the freeze-dried particles are taken out, thus obtaining the needed microbial preparation. The microbial preparation is specifically an aerobic phosphorus accumulating bacterium immobilized particle.
Example III
The embodiment is to test the adsorption effect of the microbial preparation on heavy metal ions, and specifically comprises the following steps:
respectively adding 50g of microbial preparation into heavy metal ion solutions with the same concentration, respectively detecting adsorption effect at 5min, 15min, 30min, 60min, 100min, 180min, 330min and 400min, and similarly detecting 50g of modified red pine nut shells, wherein the heavy metal ions in the embodiment are represented by heavy metal lead ions, and specific data are shown in table 3.
Embodiment four:
the embodiment is to test the purification effect of the microbial preparation on the eutrophic water body, and comprises the following specific steps:
taking eutrophic water and detecting water quality, and then dividing the eutrophic water into two parts, wherein the water quality is characterized in that: TN 7.83mg/L, TP 0.98mg/L, COD 237.8.8 mg/L, chla 235.6.235.6 mg/L. Putting the modified red pine nut shells into X-9 bacterial liquid cultured under the same condition for adsorption, fishing out the modified red pine nut shells, washing, and putting into any water body after finishing; the microbial preparation with the same quality is taken and put into another water body at the same time, and water quality detection is respectively carried out at 15d and 30d, and specific data are shown in tables 4 and 5.
Fifth embodiment:
the embodiment is a sturgeon pond application test, which comprises the following specific steps:
1kg of the microbial preparation is respectively filled into a hanging bag and the purifying unit of the invention, and two 50m microbial preparations with the same water quality and similar other conditions are screened 2 The sturgeon pond is characterized in that the hanging bag and the purifying unit are placed in different sturgeon ponds at will, water quality detection is carried out on pond water of the sturgeon ponds every five days, and specific data are shown in tables 6 and 7.
TABLE 1 TP removal Rate at different time for each Strain
TABLE 2 biomass for various strains at various times
As can be seen from tables 1 and 2, in the present test strain, X-9 had the best performance in both OD600 and TP removal, whereas in Table 2, the significant decrease in OD600 detected at 2d for X-9 was due to the inability of X-9 to release energy under adverse living conditions for X-9 to sustain its survival in adverse environments, and X-9 was selected as the target strain.
Table 3 comparison table of heavy metal ion adsorption effect
As can be seen from table 3, under the same conditions, the adsorption effect of the microbial preparation on heavy metal ions is significantly better than that of the modified red pine nut shells, and the more obvious the difference of the adsorption effect between the microbial preparation and the modified red pine nut shells is over time; the physical adsorption capacity of the modified red pine nut shells is improved through crosslinking, and the modified red pine nut shells with physical adsorption function are more beneficial to exerting physical adsorption function.
Table 4 purification effect of microbial preparation on eutrophic water
| Time/d | TN/(mg/L) | TP/(mg/L) | COD/(mg/L) | Chla/(mg/L) |
| Initial initiation | 7.83 | 0.98 | 237.8 | 235.6 |
| 15 | 2.74 | 0.32 | 100.2 | 91.9 |
| 30 | 0.90 | 0.02 | 19.6 | 11.2 |
TABLE 5 purification effect of modified red pine nut shells on eutrophic Water by adsorption of X-9
| Time/d | TN/(mg/L) | TP/(mg/L) | COD/(mg/L) | Chla/(mg/L) |
| Initial initiation | 7.83 | 0.98 | 237.8 | 235.6 |
| 15 | 3.52 | 0.48 | 133.2 | 120.1 |
| 30 | 1.6 | 0.21 | 54.7 | 44.8 |
Note that: when the modified red pine nut shells in Table 5 were fished out at 30d, part of the modified red pine nut shells were decomposed and disintegrated, while the microbial preparations in Table 4 were not significantly changed.
As can be seen from tables 4 and 5, the purification effect of the microbial preparation on the eutrophic water body under the same conditions is obviously better than that of the modified red pine nut shells for adsorbing X-9, and the partial modified red pine nut shells are decomposed and disintegrated at 30d, so that the microbial preparation is not obviously changed. Therefore, the polyethylene glycol is added to the red pine nut shells, so that the strength of the modified red pine nut shells is effectively improved, the corrosion resistance to microorganisms is improved, the modified red pine nut shells are nontoxic to organisms, and the modified red pine nut shells have good biocompatibility; the aerobic phosphorus accumulating bacteria can be better fixed on the immobilized particles through crosslinking, so that bacterial clusters are prevented from being scattered, the aerobic phosphorus accumulating bacteria are beneficial to exerting the bacterial cluster advantages, and the water purifying efficiency is improved.
Table 6 sturgeon pond bagging test results
Note that: because the water quality detected by the method is poor in 25d, the water in the sturgeon pond is subjected to additional purification treatment, and the water quality detection of 30d is not performed in the sturgeon pond bagging test.
Table 7 sturgeon pond purifying unit test results
| Time/d | TN/(mg/L) | TP/(mg/L) | COD/(mg/L) |
| Initial initiation | 0.63 | 0.18 | 20.0 |
| 5 | 0.33 | 0.04 | 13.7 |
| 10 | 0.34 | 0.03 | 11.3 |
| 15 | 0.33 | 0.02 | 11.1 |
| 20 | 0.35 | 0.02 | 11.2 |
| 25 | 0.82 | 0.11 | 16.2 |
| 30 | 1.17 | 0.21 | 26.8 |
As can be seen from tables 6 and 7, the effect of purifying the sturgeon pond water by adopting the purifying unit is obviously better than that of adopting a hanging bag, the purifying unit can move in the sturgeon pond along with water flow, the depth of the accommodating device can be changed by a lifting rope, and the different space positions of the sturgeon pond can be efficiently and conveniently purified, so that the purifying unit is used for treating the sturgeon pond water, continuous purification of the pond water quality can be ensured by only throwing the purifying unit into the pond and replacing a microbial preparation and filling a solid growth promoting agent every 25 days, the water quality of the sturgeon pond water is efficiently and conveniently ensured, and the purifying unit is simple to operate, occupies small space and has low input cost and can realize in-situ treatment.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A microbial preparation, which is characterized by comprising the following steps:
inoculating aerobic phosphorus accumulating bacteria into a synthetic culture medium, and culturing for a period of time to obtain an aerobic phosphorus accumulating bacteria liquid;
step two, rotating at 5000 r.min -1 Centrifuging for 20min, pouring out supernatant of the aerobic phosphorus accumulating bacterial liquid, and fixing the volume by using 300mL of physiological saline with the mass fraction of 0.9% to obtain a constant volume liquid;
step three, weighing the modified red pine nut shells, placing the modified red pine nut shells into the constant volume liquid for adsorption, then mixing the modified red pine nut shells with polyethylene glycol, and uniformly stirring to obtain a mixed liquid;
step four, dripping the mixed liquid into a saturated boric acid solution containing CaCl2 by using an injector with the diameter of a needle of 6-8mm, solidifying into balls, after crosslinking for a period of time, washing by using physiological saline, putting into a freeze dryer for freeze drying, and taking out freeze-dried particles to obtain the microbial preparation;
in the first step, the aerobic phosphorus accumulating bacteria are screened by adopting the following steps: taking 80mL of eutrophic water and 20g of activated sludge, adding 20mL of growth promoter, mixing, and heating at 25deg.C for r min -1 After 3 hours of culture under the condition of (2) and standing for 0.5 hour; collecting 10mL of supernatant, inoculating onto bacterial culture medium, and culturing at 25deg.C for r min -1 Is continued to be cultured for 5 days under the condition of (2); transferring 5mL culture solution, and keeping the culture medium and culture conditions unchanged; after transfer culture for 5-6 times, inoculating by streaking in a separation culture medium containing eutrophic water and activated sludgeThe method comprises the steps of continuously carrying out transfer culture on single colonies with different colors and forms for 3 times, and repeatedly separating and purifying to obtain aerobic phosphorus accumulating bacteria single colonies; respectively inoculating the obtained aerobic phosphorus accumulating bacteria single colonies into 100mL of synthetic medium for anaerobic and aerobic continuous culture for 2-3 days, measuring the OD600 and phosphorus removal efficiency of each strain, and selecting the strain with the highest OD600 and phosphorus removal efficiency as a target strain; the synthetic culture medium comprises the following components in concentration: 20g/L of activated sludge, 15g/L, naCl g/L of trypsin and 5g/L of yeast extract, and the solvent is eutrophic water body, the culture temperature is 25 ℃, and the stirring rate is 200r min -1 ；
In the third step, accurately weighing 150g red pine nut shells, crushing the red pine nut shells to be 4-6mm in size, placing the red pine nut shells in a 3L beaker, and adding 1.8L of 0.5 mol.L -1 After mixing the citric acid solutions of (2) at room temperature, 600r min -1 Stirring for 40min, transferring into a stainless steel plate, baking at 53 ℃ for 36h in a forced air drying oven, and raising the temperature to 120 ℃ for reaction for 90min to obtain modified red pine nut shells; cooling the modified red pine nut shells to room temperature, washing with deionized water until the washing liquid does not cause turbidity of the lead nitrate solution of 0.1M, and carrying out suction filtration by using suction filtration equipment; drying the modified red pine nut shells to constant weight at 60 ℃, and placing the dried modified red pine nut shells in a dryer for standby; taking out the modified red pine nut shells of 150g in the dryer, putting the modified red pine nut shells into the constant volume liquid, adsorbing for 1h, mixing the modified red pine nut shells with 200mL of polyethylene glycol with the mass fraction of 10%, and uniformly stirring to obtain a mixed liquid;
in the fourth step, the mixed liquid is dripped to CaCl with the volume of 300mL and the mass fraction of 2% by using an injector with the needle diameter of 6-8mm 2 Solidifying into balls, crosslinking for 24h, washing with physiological saline with the mass fraction of 0.9%, putting into a freeze dryer, freeze-drying at 5 ℃, and taking out freeze-dried particles to obtain the microbial preparation.
2. Use of a microbial preparation according to claim 1 for water purification, wherein the microbial preparation is placed in a purification unit which is suspended in a water body and the purification unit is thrown into the water body for water purification.
3. The use according to claim 2, wherein the purification unit comprises a floating bed for providing buoyancy, a receiving means for filling the microbial agent and a lifting rope for controlling the depth of lowering of the receiving means, one end of the lifting rope being telescopically strapped to the floating bed and the other end of the lifting rope being strapped to the receiving means.
4. The use according to claim 3, wherein the receiving means comprises a receiving means cover provided with through holes on the surface and a receiving means body provided with an inner wall and an outer wall, wall holes being provided on both the inner wall and the outer wall of the receiving means body, the inner wall and the outer wall dividing the interior of the receiving means body into an inner cavity for filling with a solid growth promoter and an outer cavity for filling with the microbial agent.
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